Expression of Antimicrobial peptide (Monodoncin) from Black Tiger Shrimp (Penaeus monodon) by Recombinant Escherichia coli

• Main Authors: Tzu-Hsuan Yang, 楊子萱
• Other Authors: Jan-Hsiung Huang
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/79892415891760510785

碩士 === 國立臺灣大學 === 微生物與生化學研究所 === 92 === Antimicrobial peptides (AMPs) were well known to play an important role in the innate host defense system of living organisms. Generally, these defensive peptides are small cationic molecules and act against invading microorganisms such as bacteria, fungi, parasites and virus. Moreover, microorganisms hardly evolve the resistance toward antimicrobial peptides but the conventional antibiotics. Recently, some studies showed that the AMPs could be successfully applied into agricultural and clinical uses. Monodoncin is an antimicrobial peptide from Black Tiger Shrimp, which contains six adjacent cysteines, proline-rich domain and one C-terminal ㄐVhelix. In this study, we introduced the inclusion body system to evaluate the production of Monodoncin in E. coli cultures, which may eliminate the degradation of target protein and reduced negative effects toward host cells aroused by target protein. We constructed the pET-Monodoncin expressing vector and introduced into the producing host E. coli BL21 (DE3). In the result of flask culture, the expression level of recombinant Monodoncin reached 13% of the total protein content (45% of the pellet) after induction by adding 0.3 mM IPTG for 10 hr. Subsequently, inclusion bodies were solubilized in 7 M urea solution at pH 12, and a one-step process of purification and renaturation were developed. Monodoncin thus obtained was 100% in purity with a recovery yield of 15.5%. The final yield was up to 13.2 mg per liter of culture. The sequence and immuno-specificity were as expectation that identical/similar to native Monodoncin according to the results from N-terminal amino acid sequencer and western blotting. The recombinant Monodoncin had been demonstrated inhibitory and bactericidial activity to Aerococcus viridans. The MIC was 176 mg/mL (24 mM). The MBC was 706 mg/mL (96 mM)。